Tissue culture offers the opportunity to investigate inborn errors of metabolism at the cellular level and to study the mechanisms by which enzymatic deficiencies give rise to abnormal phenotypes. A single defective gene can cause a cascade of effects by altering intracellular concentrations of substrates, activators and inhibitors. Changes in the cytoplasmic mileau may modulate transcription leading to further alterations in the composition of cells with effects on the phenotype. Cell culture provides a means of studying cellular metabolism in a controlled external environment unaffected by interaction with other cell types and in ways impossible to duplicate in patients. Our research goal is to elucidate the cellular and molecular mechanisms which lead to specific phenotypes in several inborn errors of metabolism as described in research projects #1-9. For example, multiple alleles that affect a membrane-associated multienzyme complex lead to a variety of phenotypes in MSUD. Genetic heterogeneity in this disorder will be studied by complementation analysis of heterokaryons (Project #4), measurement of the different enzymatic steps of BCKA decarboxylation in purified mitochondria prepared from various MSUD mutants (#1) and by purifying and characterizing the enzyme (#2) Heterozygote detection (#3) in MSUD will improve the precision of genetic counseling. Multiple enzyme deficiencies accompanying the heterogeneous group of hyperlysinemia (#6) and the putative perturbations in secondary pathways of lysine metabolism as suspected in hyperpipecolatemia (#5) will be investigated to better understand the interrelationships of interlocking pathways and to discover the enzymatic deficiencies in these diseases. Control of enzymatic reactions and substrate preferences for different pathways may be regulated by the level or availability of cofactors as suggested by studies on overproduction gout (#7). Cell to cell communication (#8) provides a mechanism for correcting the abnormal phenotype of mutant cells in heterozygotes for sex-linked recessive diseases and for coordinating and modulating changes in metabolism between interconnected cell systems. Reconstitution of cells from differentiated cytoplasts and human karyoplasts may provide insights into regulation of gene expression and permit studies on inborn errors of metabolism that are only expressed in differentiated cells (#9).